Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.
Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.
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DOI：
10.1021/bi501243y
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发表时间：
2015-01-20
期刊：
影响因子：
2.9
通讯作者：
Rothschild KJ
中科院分区：
文献类型：
--
作者：
Ogren JI;Yi A;Mamaev S;Li H;Lugtenburg J;DeGrip WJ;Spudich JL;Rothschild KJ
来自绿色鞭毛虫的视紫红质（ChRs）在哺乳动物细胞中异源表达时作为光门控离子通道发挥功能。相当大的兴趣集中在理解ChR的分子机制，以生物工程化它们的特性，用于特定的光遗传学应用，例如阐明脑回路中特定神经元的功能。虽然大多数研究都使用channelrhodopsin-2从莱茵衣藻（CrChR 2），在这项工作中，低温傅里叶变换红外差光谱应用于研究的构象变化发生在初级光跃迁的红移ChR 1从衣原体Augustae（CaChR 1）。用同位素标记的视黄醇或视黄醇类似物A2取代，定点诱变，氢-氘交换和H218 O交换用于将条带分配给视网膜发色团，蛋白质和内部水分子。在80 K的主要光跃迁的CaChR 1涉及，在相反的CrChR 2，几乎完全是一个全反式到13-顺式异构化的视网膜发色团，在细菌视紫红质（BR）的主要光跃迁。此外，显着的差异被发现的蛋白质和内部水的结构变化相比，CrChR 2，包括几个天冬氨酸/谷氨酸残基的反应，视网膜异构化。一个负酰胺II带被确定在视网膜乙烯拉伸区域的CaChR 1，这反映了沿着与酰胺I带的蛋白质骨架结构的变化在光循环早期。在CaChR 1和CrChR 2中检测到弱氢键结合的内部水的氢键强度的降低，但在CrChR 2中的谱带要宽得多，表明更不均匀的环境。涉及残基Glu 169和Asp 299（BR中Asp 85和Asp 212席夫碱抗衡离子的同系物）的突变导致Asp 299在P1形成过程中质子化的结论，并表明这些残基通过强氢键相互作用，促进质子从Glu 169转移。
Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen–deuterium exchange, and H218O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that these residues interact through a strong hydrogen bond that facilitates the transfer of a proton from Glu169.
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影响因子：
2.9
作者：
FISCHER, WB;SONAR, S;ROTHSCHILD, KJ
通讯作者：
ROTHSCHILD, KJ
影响因子：
2.9
作者：
BRAIMAN, MS;MOGI, T;ROTHSCHILD, KJ
通讯作者：
ROTHSCHILD, KJ
影响因子：
2.9
作者：
Furutani, Yuji;Sudo, Yuki;Kandori, Hideki
通讯作者：
Kandori, Hideki
影响因子：
16.2
作者：
Arenkiel, Benjamin R.;Peca, Joao;Feng, Guoping
通讯作者：
Feng, Guoping
影响因子：
2.9
作者：
BRAIMAN, MS;MOGI, T;ROTHSCHILD, KJ
通讯作者：
ROTHSCHILD, KJ